PROJECT SUMMARY/ABSTRACT Secondary bacterial infection following influenza (super-infection) is a serious clinical complication that often leads to pneumonia and death in patients. In most infection models, a single species or organism of microbial pathogen is used to induce an insult or disease in the host. However, humans are constantly exposed to a multitude of microbial pathogens simultaneously. The resulting signaling crosstalk in the immune system has largely been overlooked. An unbiased, holistic systems biology approach, therefore, is required to decipher the molecular interactions between the mammalian host, influenza virus and the bacterial pathogen. We have previously used this approach to study the immune response during single (influenza or Staphylococcus aureus) and super-infection (influenza/S. aureus). We conducted transcriptional and lipidomic analyses in samples from a mouse super-infection model. We focused our lipidomic analysis on eicosanoids because they are signaling molecules that play critical roles in the induction and resolution of inflammation. During super- infection, when compared to single infections, anti-inflammatory CYP450 metabolites, natural ligands for the nuclear receptor PPARa, were produced at a significantly higher level. We hypothesize that these lipids normally promote the physiological resolution of inflammation. However, during super-infection, CYP450 metabolites are produced at a pathological level leading to an over-activation of PPARa in innate immune cells. The activation of PPARa, in turn, compromises the anti-bacterial function of neutrophils and monocytes. The persistence of bacteria provides immune signals that amplify a feedforward loop to recruit more immune cells, thus contributing to tissue damage and eventual mortality. We will take the following approaches during single and super-infection to investigate the effects of the bioactive lipids-PPARa axis on the innate immune function and signaling. First, we will identify the transcriptional networks of infiltrating neutrophils and monocytes, the predominant cell types recruited to clear bacterial pathogens during S. aureus infection with or without prior influenza. We will further characterize the anti-bacterial function of the neutrophils and monocytes in the presence of genetic (wildtype C57/Bl6 versus Ppara?/? mice) and chemical (agonists and inhibitors against PPARa) perturbations. Using shRNA and gene editing, we will determine the genetic interactors of PPARa which collaborate to alter the transcriptional response. Second, we will determine the lipidomic landscape during the late phase of super-infection when bacterial persistence triggers further infiltration of immune cells. We will investigate whether using chemical inhibitors against PPARa and the eicosanoid metabolic pathways can alleviate the increased morbidity and mortality phenotype during super-infection. Using transcriptional and lipidomic approaches to study the bioactive lipids-PPARa axis during single and super-infection will provide significant insights into the mechanisms driving immune cross-talk during super- infections and identify novel host-directed therapeutic targets for influenza.